Relapse to drug use during abstinence is a major challenge in the treatment of drug addiction. Both human and animal studies show that relapse is often triggered and precipitated by cues previously associated with drug self-administration. Results from rodent models of drug relapse have implicated synaptic transmission from the basolateral amygdala (BLA) to the nucleus accumbens (NAc) in cue-induced cocaine seeking. The objective of this application is to explore how BLA-to-NAc synaptic transmission is re-organized following cocaine exposure and how this re-organization contributes to cue-induced cocaine seeking. Our published and preliminary results from the last funding period suggest that the BLA-to-NAc afferent undergoes a silent synapse-based re-organization and that interfering with this re-organization attenuates cue-induced cocaine seeking. Silent synapses are thought to be immature excitatory synaptic contacts that contain NMDAR receptors (NMDARs) without stable AMPA receptors (AMPARs). By recruiting/stabilizing AMPARs, these immature synapses develop into fully functional synapses, potentially resulting in new neural circuits. Thus, generation and maturation of silent synapses in the adult brain may represent a critical and profound process of synaptic and circuitry re-organization. Based on these and other results, we hypothesize that following cocaine self-administration, nascent silent synapses are generated within the BLA-to-NAc projection, and maturation of these silent synapses critically contributes to cue-induced cocaine seeking. We will test this hypothesis in the following two aims: Aim 1: Characterize cocaine-induced re-organization of BLA-to-NAc synapses We will test the hypothesis that: i) the silent synapse-based synaptic re-organization within the BLA-to-NAc afferent is initiated by the formation of new presynaptic terminals and new postsynaptic partners enriched in NR2B subunit- containing NMDARs; and ii) is completed by postsynaptic insertion of calcium-permeable AMPA receptors. We will use a combination of in vivo viral tools, molecular replacement, optogenetic manipulation, and slice electrophysiology to manipulate key proteins for axonal growth/synaptogenesis, and examine the impact of these manipulations on the generation and maturation of silent synapses within the BLA-to-NAc afferent. Aim 2: Disrupt cocaine-induced re-organization of BLA-to-NAc synapses We will develop in vivo approaches to prevent, disrupt, or reverse cocaine-induced, silent synapse-based re-organization of the BLA- to-NAc afferent, and examine the extent to which these manipulations attenuate cocaine seeking. By accomplishing the proposed experiments, we follow an innovative conceptual angle (synaptic/circuitry re-organization) to understand the neural basis underlying cue-induced cocaine seeking. Several molecular substrates characterized in this study might be targeted experimentally and clinically for attenuating cocaine relapse. As such, this proposal is highly relevant to the mission of NIDA and NIH.